The present invention relates to novel compounds useful as therapeutic agents and assay reagents. More specifically, the present invention relates to a drug targeting and especially to a novel method of delivery of therapeutically active agents to tumour cells, in particular melanoma cells.
Malignant melanoma is an important cancer with a rising incidence world-wide affecting people of all ages, including a relatively young population. Whilst progress is being made in prevention and in early diagnosis, the major problem is the difficulty of treating the disease in its disseminated state. Therapeutic agents with improved effectiveness are urgently required if the number of deaths resulting from malignant melanoma is to be reduced. Melanoma has the potential of rapid metastasis and remains a difficult neoplasm to treat. Conventional antineoplastic agents continue to be of value in the management of this malignancy but improved clinical results can be achieved if established or newly discovered agents are modified to allow them to act more selectively. The delivery of cytotoxic agents to the site of tumour cells is much desired because systemic administration of these agents often results in the killing of normal cells within the body as well as the tumour cells sought to be eliminated.
Sadly, treatment of disseminated melanoma is currently inadequate, remaining incurable. Targeted chemotherapy seems a feasible approach for this neoplasm because in the adult human melanogenesis is uniquely a property of melanocytes. Previous attempts at utilization of the melanogenic pathway to activate prodrugs have made use of the formation of reactive quinone intermediates which rely on thiol depletion for their toxic effect and initial attempts to treat melanoma in the clinic have been unsuccessful due to unfavourable pharmacokinetics, low potency and systemic toxicity.
In developing the compounds of the invention, reliance was placed on the realisation that the tyrosinase enzyme is differentially associated with certain target cells, especially melanoma cells.
Thus, tyrosinase (monophenol, 3,4-dihydroxyphenylalanine: oxygen oxidoreductase, EC 1. 14.18. 1) is generally exclusive to pigment-producing cells (melanocytes) and is frequently unregulated in melanoma. Use of the catalytic potential of tyrosinase to generate a highly toxic compound from a non-toxic substrate or xe2x80x9cprodrugxe2x80x9d has been suggested [see Riley P A (1991) Eur J Cancer, 27: 1172-1177] and so far, a number of potential melanoma prodrugs have been studied, but with limited success. Examples of such agents include analogues of tyrosine which are oxidised by tyrosinase to generate cytotoxic quinones [see e.g. Naish S, Cooksey C J and Riley P A (1988) Pig. Cell Res., 1:3 79-381; Naish S, Holden J L, Cooksey C J and Riley P A (1988) Pig. Cell Res, 1:382-385] which act through mechanisms leading to thiol depletion [see e.g. Alena F, Iwashina T, Gili A and Jimbow K (1994) Cancer Res., 54(10): 2661-2666; Alena F, Dixon W, Thomas P and Jimbow K (1995) J. Invest. Dermatol., 104(5): 792-797; Riley P A, Cooksey C J, Johnson C I, Land E J, Latter A M and Ramsden C A (1996) Eur. J. Canc. (in press)].
Initial studies indicated that compounds such as 4-hydroxyanisole held promise in targeted melanoma chemotherapy [see Morgan B D G, O""Neill T, Dewey D L, Galpine A R and Riley P A (1981) Clin. Oncol., 7:227-234], but a number of serious difficulties have been encountered in the clinic, notably the relatively limited cytotoxicity of the resulting cytotoxic quinone, necessitating the use of high serum levels of the prodrug and complications from systemic actions [see Rustin G J, Stratford M R, Lamont A, Bleehen N, Philip P A, Howells N, Wafta R R and Slack J A (1992) Eur. J. Canc., 28A, 1362-1364; Belcher H J, Nizam M and ONeill T J (1992) Br. J. Plast. Surg., 45:208-210] due to alternative metabolism, particularly hepatic and renal toxicity [see Schiller C D, Gesher A and Jheeta P (1991) Eur. J. Canc., 27:1017-1022; Stolze K and Nohl H (1991) Free Rad. Res. Comm., II:321-327. Refs. 17,18].
Therefore, in order for tyrosinase-dependent activation of cytotoxic pro-drugs is to become an effective and realistic chemotherapeutic targeting strategy for the treatment of melanoma, there has been a need for agents to be developed that produce their action by a mechanism that does not rely for its cytotoxic effect on thiol depletion.
The basis of our new approach to lethal synthesis in melanogenic cells, which to our knowledge has not hitherto been studied, is the utilization of the reductive cyclization process associated with the tyrosinase enzyme to initiate the specific intracellular release of cytotoxic agents. By this approach, we have succeeded in incorporating in a prodrug known cytotoxic compounds whose behaviour on release are well understood and which have been proven as potent inhibitors of tumour growth. The modification of the chemical structure of the active agent by incorporation in the prodrug is designed to moderate its cytotoxicity whilst it is in the form of the prodrug by diminishing its reactivity and by reduction of systemic bioavailability through effects on half-life or altered cellular uptake characteristics. The targetted release mechanism proposed is aimed at selectively liberating the drug from these constraints.
It is one aim of the present invention to produce and screen a novel category of anti-melanoma compounds. A further object is to provide novel compounds useful as assay or diagnostic agents. The approach that we adopted was based on our appreciation that the structural requirements for compounds to act as substrates for tyrosinase could be adapted to the synthesis of prodrugs.
The present invention provides a novel class of compounds which depend upon the action of tyrosinase for their conversion to desired products, including therapeutically active substances and assayable metabolites, e.g. indicator molecules.
Thus according to one aspect of the invention there is provided a pro-drug which is capable of releasing a therapeutically active agent at a desired location, characterised in that the pro-drug is a substrate for tyrosinase wherein in the presence of tyrosinase, the compound is oxidised to a quinone, which undergoes cyclisation and hydrolysis to release therapeutically active agent.
According to a further aspect of the invention there is provided a compound which is capable of conversion to an assayable substance such as an indicator molecule, characterised in that the compound is a substrate for tyrosinase wherein in the presence of tyrosinase, the compound is oxidised to a quinone, which undergoes cyclisation and hydrolysis to release said assayable substance.
More specifically, the invention provides a compound, in particular a prodrug, which is capable of releasing a therapeutically active agent or assayable substance (ThrAg) at a desired location, characterised in that the compound is a substrate for the tyrosinase enzyme and has the formula:
TyrXxe2x80x94Bxe2x80x94ThrAg* 
wherein TyrXxe2x80x94 is a residue of an optionally substituted tyrosine analogue of the structure 
wherein each of the symbols xe2x95x90Zxe2x80x94 is independently selected from xe2x95x90CHxe2x80x94, xe2x95x90Cxe2x80x94, xe2x95x90Nxe2x80x94, and xe2x80x94N+xe2x95x90O,
B represents a linking group or single bond linking TyrX and ThrAg*,
ThrAg* represents a residue of a therapeutically active agent ThrAg or a residue of an indicator molecule, and
Y represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 or a group 
n is 1, 2, 3 or 4 (preferably 1 or 2),
and either NQxe2x80x94 represents xe2x80x94Nxe2x80x94 and the dotted line represents a bond linking the nitrogen atom to the indicated ring atom or NQxe2x80x94 represents xe2x80x94NR6xe2x80x94 and the dotted line is to be ignored,
R1 and R2 independently represents hydrogen, halogen (e.g. F, Cl, Br or I) or xe2x80x94OH,
R3, R4, and R5 independently represent hydrogen, halogen (e.g. F, Cl, Br or I), C1-4 alkyl, C1-4 alkenyl, CF3, NO2, xe2x80x94OH, xe2x80x94COOH, xe2x80x94COOR, or xe2x80x94CH2OH, wherein R represents C1-4 alkyl, and
R6 represents hydrogen, halogen (e.g. F, Cl, Br or I), C1-4 alkyl, C1-4 alkenyl, CF3, NO2, xe2x80x94OH, xe2x80x94COOH, xe2x80x94CH2OH, xe2x80x94COOR, xe2x80x94OR, xe2x80x94SR or xe2x80x94SeR wherein R represents C1-4 alkyl,
and wherein in the presence of tyrosinase, the compound TyrXxe2x80x94Bxe2x80x94ThrAg* is oxidised to a quinone, which undergoes cyclization and hydrolysis to release ThrAg.
R1 and R2 are preferably selected from H and OH. R3 and R4 are preferably selected from H and xe2x80x94CH3, R5 is preferably selected from H or xe2x80x94COOH, R6 is preferably selected from H, CH3 and xe2x80x94OCH3.
Most preferably, TyrX is an optionally substituted tyrosine analogue having one of the following structures, wherein xe2x95x90Zxe2x80x94, NY, Q, B, R1, R2, R3, R4, R5, R6 and n are as defined above 
Examples of linking groups B include: 
A novel feature of the prodrugs provided in accordance with the invention is the mechanism by which the therapeutically active agent or assayable substance ThrAg is released, allowing in the case where the compound of the invention is a prodrug, targeted delivery of therapeutically active agent. This is based on attachment of the selected agent to the nitrogen atom of the structure TyrXxe2x80x94. This nitrogen atom is involved in cyclization following oxidation by the tyrosinase enzyme, resulting in formation of a group that is susceptible to hydrolysis. On tyrosinase-catalysed oxidation of the pro-drug to the quinone, cyclization produces a positively charged species which undergoes hydrolysis releasing the drug.
This mechanism is analogous to the initial phase of melanogenesis which involves the tyrosinase-catalysed oxidation of tyrosine whereby tyrosine is oxidised to the corresponding orthoquinone, which is susceptible to nucleophilic attack. A major pathway involves endogenous cyclization by reductive addition to the ring brought about by the presence of the nucleophilic amino function in the side chain to give rise to cyclodopa.
The manner in which the therapeutically active agent or assayable substance ThrAg is chemically linked to the residue of the optionally substituted tyrosine analogue TyrXxe2x80x94 will depend upon the structure of ThrAg and in particular on the availability of chemically reactive groups therein.
Thus for example, if the therapeutically active agent or assayable substance ThrAg contains a carboxy group xe2x80x94COOH, ThrAg may be linked to the optionally substituted tyrosine residue TyrXxe2x80x94 by formation of an amide linkage, TyrXxe2x80x94NH.COxe2x80x94ThrAg*. Examples of therapeutically active agents which contain carboxy groups include betulinic acid and retinoic acid.
Alternatively, if the therapeutically active agent or assayable substance ThrAg contains a hydroxy group xe2x80x94OH, ThrAg may be linked to the optionally substituted tyrosine residue TyrXxe2x80x94 by formation of an urethane linkage, TyrXxe2x80x94NH.CO.Oxe2x80x94ThrAg*.
Examples of therapeutically active agents which contain hydroxy groups include oxaliplatin derivatives, vinca alkaloid S12363, quercitin, genistein, calcipotriol and 4-hydroxy anisole.
By way of further example, if the therapeutically active agent or assayable substance ThrAg contains a primary or secondary amino group xe2x80x94NH2 or xe2x80x94NHxe2x80x94, ThrAg may be linked to the optionally substituted tyrosine residue TyrXxe2x80x94 by formation of secondary or tertiary amino linkages TyrXxe2x80x94NHxe2x80x94ThrAg* or TyrXxe2x80x94Nxe2x95x90ThrAg*. Examples of therapeutically active agents which contain primary or secondary amino groups include dacarbazine, nifedipine, staurosporin and N-methylarginine.
More generally, the abbreviation xe2x80x9cThrAgxe2x80x9d can refer to any therapeutically active agent (e.g. a cytotoxic agent), or assayable substance (e.g. an indicator molecule for use in an assay) that can be chemically modified into the pro-drug TyrXxe2x80x94Bxe2x80x94ThrAg*. Further classes of therapeutically active agents and assayable substances ThrAg include:
chemosensitising agents, i.e. chemicals that can make tumour cells sensitive to cytotoxic agents:
resistance or multidrug resistance (MDR) modifiers, i.e. chemicals that can reverse the effect of multidrug resistance expression in refractory tumour cell lines, leading to a re-sensitisation of the tumour cells to MDR drugs:
immunostimulating agents, i.e. chemicals that can prime the immune system, thus triggering an immune system against the tumour, either directly or indirectly incorporation of cytokines into a prodrug structure which can have immunomodulatory activity or other direct/indirect inhibitory actions on cancer cells
signal transduction inhibitors, i.e. chemicals that can selectively inhibit vital step/s is signalling pathways, in the normal function of the cancer cell, leading to apoptosis
differentiating agents, which can include the active metabolite of Vitamin D3 and novel analogues, retinoic acid and analogues.
repair inhibiting drugs, which can inhibit the repair of damage induced by chemotherapeutic drugs or radiation
prodrugs which when activated lead to a reduction in cellular thiol/sulphhydryl levels
prodrugs which when activated will act directly or indirectly on oncogene or tumour suppressor gene or their gene products to influence tumour growth
assayable substances or indicator molecules, including coloured or fluorescent substances such as fluorescein, raodiolabelled agents, substances which exist in differently coloured forms when free or chemically combined (e.g. dyes in colored and leuko-form), antigenic markers etc.
Prodrugs according to the invention mayl be employed either alone or in different combinations and sequences to identify the most effective drug regime.
The invention allows the selective delivery of the therapeutically active agent preferentially to those cells containing tyrosinase. The invention has the advantage of not relying on antibodies to target an enzyme to the tumour site, as used in the targeting strategy referred to as ADEPT (Antibody directed enzyme prodrug therapy).
The invention in a preferred embodiment provides a selective method for the treatment of malignant melanoma, an aggressive tumour which contains the enzyme tyrosinase. The invention may also be applicable for the treatment of other malignancies where tyrosinase is present in tumour cells, in particular breast carcinoma. The rapid expansion of the area of molecular biology and more specifically the use of retroviruses to transform cells into tyrosinase producing type, would allow a wide variety of future applications for our novel drug delivery system.
The invention can be applied for therapy to systems where selective expression of tyrosinase can successively be induced. Furthermore, the use of tyrosinase enzyme inhibitors, as small molecules or on polymer supports, can be used in conjunction with the delivery system of the invention to inactivate circulating tyrosinase, as found in melanoma patients, prior to prodrug administration. Such measures will be made to increase the degree of selectivity for melanoma cells in solid tumours.
It will be appreciated that the prodrugs provided according to the invention essentially consist of chemically modified drugs wherein the biological activity of the drug has been substantially eliminated. After administration the unmodified drug is released after the action of the enzyme tyrosinase. The release mechanism of the drug is dependent on the action of the enzyme tyrosinase, and generally occurs by a post enzyme oxidation, cyclization and hydrolysis to release the unmodified drug.
The action on tumour cells depends on the fact that certain cancer cells contain the enzyme tyrosinase, either naturally as in malignant melanoma or resulting from a spontaneous gene mutation as is reported during dedifferentiation of cancer cells or by transfection of cancer cells as in the use of retrovirus to express tyrosinase in non-pigment producing cancer cells.
The novel approach to cancer therapy according to the invention is thus based on a tyrosinase-activated drug delivery mechanism which relies on the spontaneous reductive cyclization of dopaquinone (and analogues) to render labile a potentially hydrolysable link between a nitrogen-containing group in the prodrug of formula TyrXxe2x80x94Bxe2x80x94ThrAg*. This enables the specific release of the drug in melanogenic cells, as evidenced by data presented herein.
As indicated previously a wide range of therapeutically active substances can be chemically modified to form prodrugs in accordance with the invention. Preferably, these include antineoplastic agents which are already in use in the treatment of melanoma. xe2x80x9cCombination chemotherapy for disseminated malignant melanomaxe2x80x9d. K L Abbott and G S Harman. Anti-cancer drugs 6:489-497). However, synthesis of novel prodrugs may lead to the utilization of available antineoplastic agents which are regarded as having no activity against this disease.
Examples include: 
Vinca alkaloid, S12363. An example of an agent which is too active for clinic use. Modification/incorporation of this type of molecule may provide a potent active agent being released at the tumour site. 
analoques of Dacarbazine 5-(3,3-Dimethyl-1-triazenyl)-1-H-imidazole-4-carboxamide (DTIC)
Furthermore, the therapeutically active agent can also refer to:
a chemosensitising agent, a chemical that can make tumour cells sensitive to cytotoxic agents, such as a resistance or multidrug resistance (MDR) modifier for example Nifedipine and S9788-2, the latter being a triazine derivative which contains chemical features known to be important for MDR reversing activity, planar aromatic domain and two amino groups, one having cationic charge at physiological pH. These agents can reverse the effect of multidrug resistance expression in refractory tumour cell lines, leading to a re-sensitisation of the tumour cells to MDR drugs: 
an apoptosis inducing agent, such as Staurosporin which inhibits calcium dependant phosphorylation leading to apoptosis of most types of cells. 
Staurosporin an apoptosis-inducing agent a signal transduction inhibitor, such as Quercetin or Genistein, a chemical that can inhibit the signalling pathway, such as phosphorylation of tyrosine and threonine which is essential for normal cell function. 
an immunostimulating agent, a chemical that can prime the immune system, thus triggering the immune system against the tumour, either directly or indirectly. An example of this can be by reducing the levels of Nitric oxide, by inhibiting the enzyme Nitric oxide synthetase with N-methylarginine thus removing the immunosuppressive effect on high levels of Nitric oxide, 
incorporation of cytokines (TNF-xcex1, IFN-xcex1, IFN-xcex3 or fragments of immunogenic chemicals) into a prodrug structure which can have immunomodulatory activity or other direct/indirect inhibitory actions on cancer cells,
a differentiating agent, which can include the active metabolite of Vitamin D3 and novel analogues such as Calcipotriol, retinoic acid and its analogues. 
a DNA repair enzyme inhibiting drug, such as O6-benzyl-guanine, which can inhibit the enzyme responsible for repairing nuclear DNA damage induced by chemotherapeutic drugs or radiation,
a chemical, such as p-hydroxy anisole which when activated to the quinone leads to a reduction in cellular thiol/sulphhydryl levels rendering cells more susceptible to damage by chemical agents. Use of analogues of Buthionine sulfoximine (BSO) a glutamyl cysteine synthetase to reduce the level of cellular glutathinone synthesis (Kable et al [1989] Cancer Res, 49: 2327-2331) 
an antisense molecule, which when activated will act directly or indirectly on oncogene or tumour suppressor gene or their gene products to influence tumour growth.
The prodrugs of the invention which can be synthesized by the initial formation of the activated mixed anhydrides of the selected agents which are then converted to the potential prodrug by nucleophilic attack by tyrosine. The general systemic cytotoxicity of the prodrugs will be diminished by incorporation of the reactive functional groups in the covalent bond to the tyrosine.
The invention further provides the use of the compounds of the invention in which are capable of conversion to an assayable substance in diagnostic and assay procedures. These include assay procedures for detecting the presence of tyrosinase enzyme in tissue samples and body fluids. In such assays, the sample to be assayed may be contacted with the compound of the invention and following an appropriate incubation period, the release of assayable substance detected. Such procedures are of particular use in the diagnosis of disease states where levels of tyrosinase enzyme are altered, as in melanomas associated with elevated tyurosinase levels.